Peptidomic Workflow Applied to Cerebrospinal Fluid Analysis

  • Rustam H. Ziganshin
  • Sergey I. Kovalchuk
  • Igor V. Azarkin
Part of the Methods in Molecular Biology book series (MIMB, volume 2044)


Proteo-peptidomic profiling of biofluids is used to identify disease biomarkers and to study molecular mechanisms of pathology development. Previously, we studied changes in cerebrospinal fluid (CSF) and blood plasma associated with Guillain-Barre syndrome (GBS)—a rare and severe disorder of the peripheral nervous system with an unknown etiology. Here, we describe the workflow for the analysis of endogenous peptides from CSF. The procedure covers sample preparation, liquid chromatography-mass spectrometry (LC-MS) analysis, and bioinformatics analysis and allows identification of more than 1100 peptides from 181 protein groups in ~3 h from a single CSF sample derived from non-neurological, non-oncological patients.

Key words

Endogenous peptides Peptide isolation Peptidome Cerebrospinal fluid LC-MS SDB-RPS StageTips 


  1. 1.
    Illes S (2017) More than a drainage fluid: the role of CSF in signaling in the brain and other effects on brain tissue. Handb Clin Neurol 146:33–46. Scholar
  2. 2.
    Ziganshin RH, Ivanova OM, Lomakin YA, Belogurov AA Jr, Kovalchuk SI, Azarkin IV, Arapidi GP, Anikanov NA, Shender VO, Piradov MA, Suponeva NA, Vorobyeva AA, Gabibov AG, Ivanov VT, Govorun VM (2016) The pathogenesis of the demyelinating form of Guillain-Barre syndrome (GBS): proteo-peptidomic and immunological profiling of physiological fluids. Mol Cell Proteomics 15(7):2366–2378. Scholar
  3. 3.
    Lowenthal MS, Mehta AI, Frogale K, Bandle RW, Araujo RP, Hood BL, Veenstra TD, Conrads TP, Goldsmith P, Fishman D, Petricoin EF 3rd, Liotta LA (2005) Analysis of albumin-associated peptides and proteins from ovarian cancer patients. Clin Chem 51(10):1933–1945. Scholar
  4. 4.
    Mehta AI, Ross S, Lowenthal MS, Fusaro V, Fishman DA, Petricoin EF 3rd, Liotta LA (2003) Biomarker amplification by serum carrier protein binding. Dis Markers 19(1):1–10CrossRefGoogle Scholar
  5. 5.
    Chertov O, Biragyn A, Kwak LW, Simpson JT, Boronina T, Hoang VM, Prieto DA, Conrads TP, Veenstra TD, Fisher RJ (2004) Organic solvent extraction of proteins and peptides from serum as an effective sample preparation for detection and identification of biomarkers by mass spectrometry. Proteomics 4(4):1195–1203. Scholar
  6. 6.
    Tucholska M, Florentinus A, Williams D, Marshall JG (2010) The endogenous peptides of normal human serum extracted from the acetonitrile-insoluble precipitate using modified aqueous buffer with analysis by LC-ESI-Paul ion trap and Qq-TOF. J Proteome 73(6):1254–1269. Scholar
  7. 7.
    Williams D, Ackloo S, Zhu P, Bowden P, Evans KR, Addison CL, Lock C, Marshall JG (2010) Precipitation and selective extraction of human serum endogenous peptides with analysis by quadrupole time-of-flight mass spectrometry reveals posttranslational modifications and low-abundance peptides. Anal Bioanal Chem 396(3):1223–1247. Scholar
  8. 8.
    Orvisky E, Drake SK, Martin BM, Abdel-Hamid M, Ressom HW, Varghese RS, An Y, Saha D, Hortin GL, Loffredo CA, Goldman R (2006) Enrichment of low molecular weight fraction of serum for MS analysis of peptides associated with hepatocellular carcinoma. Proteomics 6(9):2895–2902. Scholar
  9. 9.
    Tirumalai RS, Chan KC, Prieto DA, Issaq HJ, Conrads TP, Veenstra TD (2003) Characterization of the low molecular weight human serum proteome. Mol Cell Proteomics 2(10):1096–1103. Scholar
  10. 10.
    Wu J, An Y, Pu H, Shan Y, Ren X, An M, Wang Q, Wei S, Ji J (2010) Enrichment of serum low-molecular-weight proteins using C18 absorbent under urea/dithiothreitol denatured environment. Anal Biochem 398(1):34–44. Scholar
  11. 11.
    Kawashima Y, Fukutomi T, Tomonaga T, Takahashi H, Nomura F, Maeda T, Kodera Y (2010) High-yield peptide-extraction method for the discovery of subnanomolar biomarkers from small serum samples. J Proteome Res 9(4):1694–1705. Scholar
  12. 12.
    Greening DW, Simpson RJ (2010) A centrifugal ultrafiltration strategy for isolating the low-molecular weight (<or=25K) component of human plasma proteome. J Proteome 73(3):637–648. Scholar
  13. 13.
    Gil GC, Brennan J, Throckmorton DJ, Branda SS, Chirica GS (2011) Automated analysis of mouse serum peptidome using restricted access media and nanoliquid chromatography-tandem mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci 879(15-16):1112–1120. Scholar
  14. 14.
    Chen H, Liu S, Yang H, Mao Y, Deng C, Zhang X, Yang P (2010) Selective separation and enrichment of peptides for MS analysis using the microspheres composed of Fe3O4@nSiO2 core and perpendicularly aligned mesoporous SiO2 shell. Proteomics 10(5):930–939. Scholar
  15. 15.
    Hu L, Boos KS, Ye M, Wu R, Zou H (2009) Selective on-line serum peptide extraction and multidimensional separation by coupling a restricted-access material-based capillary trap column with nanoliquid chromatography-tandem mass spectrometry. J Chromatogr A 1216(28):5377–5384. Scholar
  16. 16.
    Zheng X, Baker H, Hancock WS (2006) Analysis of the low molecular weight serum peptidome using ultrafiltration and a hybrid ion trap-Fourier transform mass spectrometer. J Chromatogr A 1120(1-2):173–184. Scholar
  17. 17.
    Rai AJ, Gelfand CA, Haywood BC, Warunek DJ, Yi J, Schuchard MD, Mehigh RJ, Cockrill SL, Scott GB, Tammen H, Schulz-Knappe P, Speicher DW, Vitzthum F, Haab BB, Siest G, Chan DW (2005) HUPO plasma proteome project specimen collection and handling: towards the standardization of parameters for plasma proteome samples. Proteomics 5(13):3262–3277. Scholar
  18. 18.
    Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2(8):1896–1906. Scholar
  19. 19.
    Tian R, Zhang H, Ye M, Jiang X, Hu L, Li X, Bao X, Zou H (2007) Selective extraction of peptides from human plasma by highly ordered mesoporous silica particles for peptidome analysis. Angew Chem Int Ed Engl 46(6):962–965. Scholar
  20. 20.
    Ziganshin R, Arapidi G, Azarkin I, Zaryadieva E, Alexeev D, Govorun V, Ivanov V (2011) New method for peptide desorption from abundant blood proteins for plasma/serum peptidome analyses by mass spectrometry. J Proteome 74(5):595–606. Scholar
  21. 21.
    Ziganshin RH, Ryabinin VV, Azarkin IV, Govorun VM, Ivanov VT (2018) Optimization of conditions for blood plasma peptidome analysis. Russ J Bioorg Chem+ 44(3):289–295. Scholar
  22. 22.
    Kovalchuk SI, Jensen ON, Rogowska-Wrzesinska A (2018) FlashPack: fast and simple preparation of ultra-high performance capillary columns for LC-MS. Mol Cell Proteomics. Scholar
  23. 23.
    Ma B, Zhang K, Hendrie C, Liang C, Li M, Doherty-Kirby A, Lajoie G (2003) PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid Commun Mass Spectrom 17(20):2337–2342. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Rustam H. Ziganshin
    • 1
  • Sergey I. Kovalchuk
    • 1
  • Igor V. Azarkin
    • 1
  1. 1.Shemyakin-Ovchinnikov Institute of Bioorganic ChemistryMoscowRussian Federation

Personalised recommendations